INTRODUCTION:

Alzheimer's disease (AD) is a slow progressive neurodegenerative disease of the brain that is characterized by impairment of memory and eventual disturbances in reasoning, planning, language, and perception. Memory is the ability of an individual to record sensory stimuli, events, information, etc., retain them over short or long periods of time and recall the same at a later date when needed, Poor memory, lower retention and slow recall are common problems in today’s stressful and competitive world¹.

Learning and memory are two fundamental cognitive functions that confer us the ability to accumulate knowledge from our experiences.

Learning refers to the acquisition of any new information about the events occurring in surroundings and subsequent retrieval of this information is referred to as memory. Memory is one of the most complex functions of the brain and ultimately involves multiple neuronal pathways and neurotransmitters. Impairment of memory is an organic brain disorder defined as loss of intellectual ability of sufficient severity to interfere either with occupational functioning, usual social activities or relationship of a person in the absence of gross clouding of consciousness or motor involvement, multiple brain structures hippocampus, striatum, amygdale are important for different types of memory.²

Dementia is defined as the significant loss of cognitive abilities severe enough to interfere with social functioning. It can result from various diseases that cause damage to brain cells. There are many different types of dementia, each with its own cause and symptoms. For example, vascular dementia is caused by decreased blood flow to a part of the brain, as caused by a stroke. Dementia may also be present in patients with Parkinson’s disease and hydrocephalus. AD is the most common form of dementia, caused by the build-up of beta myeloid plaques in the brain.³

Herbal remedies which were used traditionally now significantly documented for the safety profile and as a therapy for some of the pathological conditions. From the last decade synthetic drugs have been combined with herbs which show some promising results. SUMENTA, a licensed polyherbal formulation contains the extracts of medicinal plants, but no scientific report available for its nootropic activity. Its efficacy in AD and dementia has not been validated using modern scientific parameters. Hence, the present study was aimed to investigate nootropic activity of licensed polyherbal formulation “SUMENTA”. The main aim of this work was to investigate the nootropic activity of polyherbal formulation and the evaluation will conducted using various animal models.

MATERIALS AND METHOD

Sumenta tablets is a poly herbal formulation manufactured by Charak Pharma Baddi, Solan, Himachal pradesh (H.P) and was purchased from the Dakshina Karnataka district, mangalore. It is preserved in the departmental library for future reference .Piracetam from Micro Labs India, scopolamine from Cadila Health Care India Ahmedabad

Animal selection:

Swiss albino mice having young (18gm i.e 8 weeks) and aged (25gm i.e 28 weeks) were used for the study. The mice were inbred in the central animal house of the Department of Pharmacology, Karavali College of Pharmacy, Mangalore, under suitable conditions of housing, temperature, ventilation and nutrition were used for the experiments. They were kept in clean dry cages week before the beginning of the experiment to acclimatize with the experimental conditions. The animals were fed with standard pellet diet _{and distilled water add libitum was maintained at 210C-230C under a constant 12hrs light}and dark cycle. The animal care and experimental protocols were in accordance with CPCSEA /IAEC.

Preparation of test solution:

The tablets was crushed in to powder, a small quantity of water is added to make a smooth suspension, triturated with mortar and pestle, a pinch of Tween 80 is added to make a uniform suspension.

Acute oral toxicity study.⁴

The dose selection of Sumenta were based on acute toxicity studies. The acute toxicity of formulation was determined by using albino mice (20-30gm) those maintained under standard husbandry conditions. The animals were fasted three hours prior to the experiment. Animals were administered with single dose of the solution of Sumenta tablet and observed for its mortality up to 48 hr study period (short term toxicity). Based on the short term toxicity profile, the next dose was determined as per OECD guidelines NO 425. From the LD₅₀ dose 1/20, 1/10 and 1/5^th doses were selected and considered as low, medium and high dose respectively and used in the entire study.

A. TEST FOR LOCOMOTOR ACTIVITY.^5,6

1. Locomotors function test in young mice

Method: Adult Swiss albino mice (young mice) divided in to five groups and each group containing six animals are fasted overnight, prior to the test but water was supplied ad libitum. The details of experimental protocol given below.

Group I: Control group:

Distilled water (10ml/kg) was administered p.o for 8 days. After 90 min of administration, no of locomotion on 8^th day and 9^th day, was recorded.

Group II: Standard group:

Piracetam (200mg/kg) was administered p.o for 8 days. After 90 min of administration, no of locomotion on 8^th day and 9^th day, was recorded.

Group III:

PHFS (250mg/kg) was administered p.o for 8 days.After 90 min of administration , no of locomotion on 8^th day and 9^th day, was recorded.

Group IV:

PHFS (500mg/kg) was administered p.o for 8 days. After 90 min of administration , no of locomotion on 8^th day and 9^th day, was recorded.

Group V:

PHFS (250mg/kg) was administered p.o for 8 days. After 90 min of administration , no of locomotion on 8^th day and 9^th day, was recorded.

2. Locomotors function test in aged mice

Method: Adult Swiss albino mice (aged mice) divided in to five groups and each group containing six animals are fasted overnight, prior to the test but water was supplied ad libitum. The details of experimental protocol given below.

Group I: Control group:

Distilled water (10ml/kg) was administered p.o for 8 days. After 90 min of administration, no of locomotion on 8^th day and 9^th day, was recorded.

Group II: Standard group:

Piracetam (200mg/kg) was administered i.p for 8 days. After 90 min of administration, no of locomotion on 8^th day and 9^th day, was recorded.

Group III:

PHFS (250mg/kg) was administered p.o for 8 days. After 90 min of administration , no of locomotion on 8^th day and 9^th day, was recorded.

Group IV:

PHFS (500mg/kg) was administered p.o for 8 days. After 90 min of administration, no of locomotion on 8^th day and 9^th day, was recorded.

Group V:

PHFS (750mg/kg) was administered p.o for 8 days. After 90 min of administration, no of locomotion on 8^th day and 9^th day, was recorded.

Experimental Procedure:

Albino mice of either sex were divided in to five groups of six mice in each group. Animals are fasted over night prior to the test but water was supplied ad libitum. The animals presented in each group was treated with drugs as above manner. One hour after the above treatment, each mouse was placed individually in actophotometer for a period of 10 min and locomotor activity was measured in terms of scores.

B. HEBB’S-WILIAM MAZE¹

The Hebb’s-William maze consists of completely enclosed rectangular box with an entry and a reward chamber appended at opposite ends. The box is partitioned with wooden slats into blind passages leaving just one twisting corridor leading from the entry to the reward chamber.

Hebb’s William maze is used for studying learning, memory and reasoning in animals. The clever the rat, the more quickly it is able to make use of past experience and therefore more quickly it learns its way out in the maze. In the present study, we evaluated the effects of SUMENTA on learning, memory and reasoning model using the Hebb’s William maze in mice.

The learning assessment for control and drug treated animals was conducted at end of drug treatment under zero watt red colored bulb so as to minimize the nocturnal cycle disturbances. Before the training all the animals were familiarized with Hebb’s William maze for a period of 10 min. From 1st -3rd day, the rats received four consecutive trials of training per day in the maze. In each trial the rat was placed in the entry chamber and the timer was activated as soon as the rat leaves the chamber. The time taken for the mice to reach the reward chamber (T L in minutes) was taken as the learning score of the trial. The average of four trials was taken as the learning score for the day. Lower scores of assessment indicate efficient learning while higher scores indicate poor learning in animals. Retention of this learned-task (memory) was examined 96 hr after the last day trail (i.e., 9th day). Significant reduction in TL value of retention indicated improvement in memory.⁷

I. Transfer latency test in young mace:

Method: Adult Swiss albino mice (young mice) divided in to six groups and each group containing six animals are fasted overnight, prior to the test but water was supplied ad libitum. The details of experimental protocol given below.

Group I: Control group:

Distilled water (10ml/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day TL (sec) are recorded and again after 24 hr i.e. on 9^th day.

Group II: Standard group:

Piracetam (200mg/kg) was administered p.o for 8 days. After 90 min of administration on 8^thday TL (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group III:

Scopolamine (0.4 mg/kg) was administered p.o on 8^th day. After 90 min administration on 8^th day, TL (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group IV:

PHFS (250mg/kg) was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mints before the administration of PHFS. After 90 min administration on 8^th day TL (sec) was recorded and again after 24 hr i.e. 9^th on day.

Group V:

PHFS (500mg/kg) was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mints before the administration of PHFA. After 90 min administration on 8^th day TL (sec) was recorded and again after 24 hr i.e. 9^th on day.

Group VI:

PHFS (750mg/kg) was administered p.o for 8 days. Scopolamine (0.4mg/kg) was also administered i.p 45 mints before the administration of PHFS. After 90 min administration on 8^th day TL (sec) was recorded and again after 24 hr i.e. 9^th on day.

1. Transfer latency test in aged mice:

Group I: Control group:

Distilled water (10ml/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day TL (sec) are recorded and again after 24 hr i.e. on 9^th day.

Group II: Standard group:

Piracetam (200mg/kg) was administered p.o for 8 days. After 90 min of administration on 8^th day TL (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group III:

PHFS (250mg/kg) was administered p.o for 8 days. Scopolamine (1.0mg/kg) was also administered i.p 45 mts before the administration of PHFS. After 90 min administration on 8^th day, TL (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group IV:

PHFS (500mg/kg) was administered p.o for 8 days. Scopolamine (1.0mg/kg) was also administered i.p 45 mts before the administration of PHFS. After 90 min administration on 8^th day, TL (sec) was recorded and again after 24 hr i.e. on 9^th day.

Group V:

PHFS (750mg/kg) was administered p.o for 8 days. Scopolamine (1.0mg/kg) was also administered i.p 45 mts before the administration of PHFS. After 90 min administration on 8^th day, TL (sec) was recorded and again after 24 hr i.e. on 9^th day.

Experimental method

Significant reduction in TL value of retention indicated improvement of memory. The time taken by the animal (Learning score) to reach the reward chamber from the entry chamber in all the doses of Sumenta (250,500 and 750 mg/kg, p.o) treated animals was reduced on day 1, 2, and 3, when compared to respective control groups indicating significant improvement in memory. Even there was significant reduction in TL value of retention (memory) on day 7th at all the doses of Sumenta (250, 500 and 750 mg/kg, p.o) .¹

Statistical analysis.

Data were presented as mean ± Standard Error of Mean (SEM). One-way Analysis Of Variance (ANOVA), followed by Dunnet’s multiple comparison test. For all test probability 0.05 or less was accepted as significance.

ANOVA (Analysis of variance).

In statistics, analysis of variance is a collection of statistical models and their associated procedures, in which the observed variance is partitioned into components due to different explanatory variables. In its simplest form ANOVA gives a statistical test of whether the means of several groups are all equal and therefore generalize Dunnett’s multiple comparison tests to more than two groups.

RESULT :

A. ACUTE ORAL TOXICITY STUDY:

The mice treated with 5000mg/kg, p.o dose of Sumenta tablet exhibited normal behavior, without any signs of passivity, stereotypy and vocalization. The motor activity and secretary signs were also normal and no sign of depression. Further 5000mg/kg body weight did not produce any mortality. So 1/20^th and 1/10^th doses of 250mg/kg were selected as low, and 750 mg/kg were selected as high doses and were tested in the present study to explore nootropic activity.

LOCOMOTOR FUNCTION TEST:

Locomotor activity of control as well as test groups were evaluated by using actophotometer. There was no significant increase in locomotor activity in the test group compared to control in both young as well as aged mice.

The animals treated with piracetam and scopolamine showed a significant increase in the locomotor activity in young as well as aged mice compared to control group, which was more significant in young mice (P≤0.001) compared to aged mice (P≤0.01)

B. HEBB’S-WILLIAM MAZE

The Hebb’s-William maze model is to evaluate learning and memory in mice. In the present study the aged mice showed a slight increase in TL on 8^th day of the treatment, indicating impairment in learning and memory (ageing induced amnesia). Piracetam (200mg/kg) pre treatment for 8 days showed a significant decrease in the transfer latency in both young as well as aged mice indicating improvement in both learning and memory. But it was significant (P<0.001) compared to control in young mice. Scopolamine (1.0 mg/kg) shown a significant decrease in TL in both the young as well as aged mice compared to control, indicating impairment of memory. On the 9^th day 750 PHFS shown significant reduction in TL time when comparing with control

In exteroceptive behavioural model in young mice, low dose of PHFS (250mg/kg p.o) show slightly decreased TL on 8^th and 9^th day in young mice, when compared to control groups. Medium and higher dose of PHFS (500 mg/kg and 750 mg/kg) show improved learning and memory of young mice reflected by decrease in TL on 8^th and 9^thday, when subjected to EPM test. PHFS pre-treatment for 8 days protected the young as well as the old mice old mice against scopolamine induced amnesia.

In interoceptive model in young mice scopolamine (0.4 mg/kg i.p) slightly increase the, TL in young mice on 8^th and 9^th day as compared to control, indicating impairment of both learning and memory. In lower dose of PHFS (250 mg/kg p.o) show slight decrease in TL, medium (500mg/kg p.o) and higher (750 mg/kg p.o) dose also produce decrease in TL in both 8^th and 9^th day.

In introceptive model in aged mice (Table 1) low dose of PHFS (250mg/kg p.o) slightly decrease TL on 8^th and 9^th day. But medium and higher dose produce significantly decrease TL in both 8^th and 9^th day.

Table 1 Locomotor function test in young mice.

Sl. No	Treatment	No of locomotion on 8^thday	No of locomotion on 9^thday
1	Control	203.00±0.57	200.30±0.98
2	Piracetam	201.50±0.76	197.00±0.516**
3	PHFS 250	198.50±0.42 ***	198.30±0.55
4	PHFS 500	200.50±0.56	198.50±0.67
5	PHFS 750	201.20±.96	190.30±.42 ***

Data is expressed as a mean ±S.E.M for n=6, Statistical analysis by one-way ANOVA followed by Dunnett’s t test significance at *P<0.05, **P<0.01, ***P<0.001.

Table 2; Locomotor function test in aged mice.

Sl.no	Treatment	No of locomotion on 8^th day	No of locomotion on 9^th day
1	Control	178.50±0.42	171.00±1.93
2	Piracetam	179.70±1.33**	167.70±0.95***
3	PHFS 250 gm	179.20±0.60	170.00±1.14
4	PHFS 500 gm	179.00±0.64**	170.00±.68
5	PHFS 750 gm	180.00±0.91	170.73±0.84***

Data is expressed as a mean ±S.E.M for n=6, Statistical analysis by one-way ANOVA followed by Dunnett’s t test significance at *P<0.05, **P<0.01, ***P<0.001.

Table 3:Effect of PHFS on Hebb’S-William maze in young mice

Sl. No.	Treatment	TL (sec) on 8^th day	TL (sec) on 9^th day
1	Control	239.70 ±0.61	236.70 ±1.17
2	Piracetam	343.20 ± 0.79***	335.20 ± 0.79***
3	Scopolamine	182.30 ± 1.45***	146.20 ± 1.67
4	PHFS 250 mg	149.30 ± 1.62	171.50 ± 1.33
5	PHFS 500 mg	148.70 ± 2.12**	153.20 ± 1.93***
6	PHFS 750 mg	139.7 ± 2.12***	137.7 ± 0.66**

Data is expressed as a mean ±S.E.M for n=6, Statistical analysis by one-way ANOVA followed by Dunnett’s t test significance at *P<0.05, **P<0.01, ***P<0.001.

Table4: Effect of PHFS on Hebb’S –William maze in aged mice

Sl. no	Treatment	TL (sec) on 8^th day	TL (sec) on 9^th day
1	Control	227.20 ±0.60	227.20 ±1.04
2	Piracetam	285.80 ± 1.38***	284.70 ± 1.58***
3	PHFS 250 mg	147.80 ± 0.85**	148.80 ± 1.49**
4	PHFS 500 mg	144.70 ± 1.33**	144.70 ± 1.33
5	PHFS 750 mg	137.33 ± 1.93***	138.33 ± 2.33***

Data is expressed as a mean ±S.E.M for n=6, Statistical analysis by one-way ANOVA followed by Dunnett’s t test significance at *P<0.05, **P<0.01.

DISCUSSION:

Alzheimer’s disease is a very complex disorder which was described by Alois Alzheimer more than 100 years ago. Although its underlying pathology is still not entirely clear, research has accumulated a wealth of information on different mechanisms that account for one or the other aspect of the disease. Thus, an important target for the development of new treatment approaches was identified which is still the basis of many research projects.⁸

The concept and definition of a nootropic drugs was first proposed in 1972 by C.E. Guirgea and coined the term nootropic from italic words ‘noos’(mind)and tropein (to turn towards) to mean enhancement of learning and memory.⁹

Nootropics are the drugs better known as smart drugs, memory cognitive enhancers, suppliments, functional foods or neutraceuticals that improve mental functions, such as cognitive attention, concentration, motivation, intelligence and memory. It is thought that these drugs enhance memory by increasing brains supply of neurochemicals like neurotransmitters, enzymes and hormones that improve oxygen supply to brain or stimulating nerve growth.¹⁰

Sumenta tablets containts Bacopa monnieri, Withania somnifera, Nardostachys jatamansi, Celastrus paniculatus, Aegle marmelos, Emblica officinanis, Terminilia arjuna, Acorus catamus, evolves alsinoides, valerian wallichi,

Different types of mechanisms are proposed to different types of drugs and all these drugs however influence cholinergic function by improving the uptake of choline and facilitate the production and turnover of acetylcholine and produce action at both muscarinic and nicotinic receptors. In human individuals a drastic decline in Ach receptor are reported with aging drastic. Nootropic drug piracetam was reported to elevate the frontal cortex density of Ach receptors by 30-40% and this restore the Ach in the brain.⁸

Exact mechanism for memory dysfunction is not clear but some of the evidences like energy dependent inhibition of neuronal membrane function, formation of oedema, opening of voltage dependent Ca²⁺ channels (LandN type) and activation of NMDA receptors, Ca²⁺ influx with subsequent activation of NMDA receptor, Ca²⁺ influx with subsequent activation of Ca²⁺/ calmodulin dependent nitric oxide NO syntheses and NO are demonstrated. This NO may react with superoxide anion to produce highly toxic peroxy nitrite, which is responsible for energy depletion. All the above mentioned are responsible for the impairment of memory. Still a controversial but a predominant role of cholinergic mechanisms long been stressed in learning and memory process. The rate of the central cholinergic system is well established in memory and deficiency of this is implicated in deficit in memory. Though a very good number of other receptor systems are also now reported to involve in the behavioural expression in dementia in animals and human beings as well and the role of these neurotransmitter system cannot be ignored.¹¹

In locomotors function test, there was no significant increase in locomotors activity in the test group compared to control in both young as well as aged mice. The animals treated with piracetam and scopolamine showed no significant changes in the locomotors activity in young as well as aged mice compared to control group.

In Hebb’s William maze test, the test drug alone did not show significant decrease in the transfer latency in young and aged groups of mice compared to control. The animals treated with piracetam increase TL and scopolamine alone decrease TL compared with the control group in aged mice. The test drug showed that significantly increase the TL value in both young and aged group compared with normal group.

The literature review on screening on screening of herbs in AD revealed that constituents like flavonoids, tannins, alkaloids and saponins reported to be responsible for anti Alzheimer’s activity. The active ingredients of Sumenta contains flavonoids, tannins, alkaloid, saponins and trace elements.

CONCLUSION:

The nootropic activity of the extract of Sumenta were evaluated by locomotors function test in mice and, Hebbs William maze. The results obtained in different kinds of test showed that the polyherbal formulation Sumenta has shown anti Alzheimer’s activity. No mortality or behavioural abnormality recorded in mice even at the highest dose level 5000mg/kg and 7000mg/kg tested for LD₅₀ studies. In the study The animal treated with piracetam and test drug showed no significant action on the locomotors activity in young as well as aged mice in locomotors function test compared to control group. This revealed that Sumenta has no sedative action. The test drug showed that significantly increase the TL value in both young and aged group compared with normal group.

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Received on 16.04.2015 Modified on 23.05.2015

Res. J. Pharmacology & P’dynamics. 7(2): April- June 2015; Page 61-67

DOI: 10.5958/2321-5836.2015.00013.0